The ability of complement to recognize ligand/anti-ligand binding events on the surface of lipid vesicles, leading to complement-mediated vesicle lysis, has been exploited in immunoassays. Yasuda, et al have described a simple method for measuring anti-glycolipid antibody by reacting the antibody with complement and liposomes containing surface glycolipid, where complement-mediated cell lysis and release of a fluorogenic reporter from the liposomes produces increased fluorescence in the assay medium (reference 1). Similar types of liposome immunoassays applicable to ganglioside GM.sub.2 antigen (reference 2), and to Forsmann, and blood group A-reactive gangliolipids (reference 3) have been reported.
Immunoassay systems involving lysable lipid membrane vesicles provide important potential advantages in diagnostic test systems. One advantage is that the ligand/anti-ligand binding reaction, and the measurement of released reporter from lysed cells, can be performed in the same assay mixture. This single-mixture assay, which is referred to as a homogeneous assay, contrasts with "solid-phase" enzyme or fluorescent immunoassays which involve the steps of (1) binding a ligand-reporter complex to a solid surface in the presence of analyte in one mixture, (2) separating the solid and liquid phases, and (3) measuring bound or unbound reporter levels in the solid or liquid phases, respectively. Homogeneous cell-lysis immunoassays also have the potential for high assay sensitivity, since relatively few ligand/anti-ligand binding events on the vesicle surface can lead to the release or expression of a large number of reporter molecules.
Several types of encapsulated reporter compounds have been used in lipid-vesicle reagents in immunoassays of this type. Chromogenic compounds such as hemoglobin have been used, e.g., in red blood cell vesicle reagents, and fluorogenic reporters have also been described, as indicated above. U.S. Pat. No. 3,887,698 to McConnell, et al. discloses a liposome immunoassay test system in which the difference in electron paramagnetic resonance spectra between encapsulated and released nitroxide reporter provides a measure of complement-mediated liposome lysis. U.S. Pat. No. 4,235,792, to Hsia, et al. and U.S. Pat. No. 4,342,826, to Cole disclose liposome immunoassay systems in which complement-mediated cell lysis is evidenced by the expression of liposome-encapsulated enzymes.
Among the several types of reporter molecules which can be used, encapsulated enzymes offer a number of advantages over smaller chromogenic, fluorogenic or paramagnetic reporter molecules. Unlike smaller reporter molecules, enzymes show very little leakage on storage, or release due to non-specific lysis in the presence of complement. Therefore, background levels in the immunoassay can be kept quite low. Many enzymes can participate in reactions which lead to detectable color changes, allowing an assay to be monitored visually, and, in any case, without the type of relatively expensive detecting apparatus which is required for determining released fluorogenic or paramagnetic reporters. Another important advantage of enzymes is the multiplication in sensitivity which is possible because of the high turnover of each enzyme molecule.
Despite the attractiveness of a lipid-vesicle immunoassay having encapsulated enzymes, difficulties in providing a satisfactory enzyme-encapsulated vesicle reagent have been encountered. Some enzymes, such as alkaline phosphatase, are present at relatively high levels in serum, and are therefore generally unsuitable for use in an assay system for determination of a serum analyte. Another major problem encountered is the difficulty in producing a vesicle reagent that has both (1) a high specific activity of an encapsulated enzyme, and (2) a substantially oligolamellar lipid bilayer structure. Experiments conducted in support of the present invention, and reported below, indicate that complement-mediated vesicle lysis results in much higher levels of enzyme released from oligolamellar vesicles--defined as having one or a few lipid bilayer shells--than from multilamellar vesicles. The greater enzyme release, in turn, permits a considerably more sensitive homogeneous immunoassay.
A number of methods for producing large oligolamellar vesicles based on injecting a solution of lipid into an aqueous solution have been described (references 4-6). These methods give relatively poor encapsulation efficiencies, and therefore are impractical for use in producing a reagent having encapsulated enzyme with high specific activity. A reverse evaporation phase method for producing oligolamellar vesicles under conditions of relatively high encapsulation efficiency has been described (reference 7). Heretofore, however, inactivation of enzymes by exposure to the organic solvent or solvents and to sonication used in the method have limited the use of the method in forming vesicles having encapsulated enzymes.
Another limitation of encapsulated-enzyme liposomes which has been encountered is the difficulty in storing an enzyme-encapsulated liposome reagent without serious loss of enzyme activity over relatively short storage periods, such as a few weeks.